In many applications, it is desirable to target cells and tissue for localized heating or imaging. The therapeutic effects range from the destruction of cancerous cells and tumors, to the therapeutic or cosmetic removal of benign tumors and other tissue. Techniques which effect precise localized heating and illumination would allow one to enjoy therapeutic and diagnostic benefits, while minimizing the collateral damage to nearby cells and tissue. It is desirable that such techniques be amenable to both in vitro and in vivo therapeutic and diagnostic applications of induced hyperthermia and imaging, respectively, of cells and tissue.
A potentially useful in vivo application of such a technique would be in cancer reatment. For example, metastatic prostate cancer is a leading cause of mortality in American men. Estimates indicate that greater than one in every eleven men in the U.S. will develop prostate cancer. Accurate determination of the extent of local disease is often difficult. Methods for accurately detecting and imaging localized prostate disease are greatly needed. In addition, localized prostate cancer is generally treated with either radical prostatectomy or radiation therapy. Both of these procedures are plagued by significant morbidity. Minimally invasive treatment strategies with low associated morbidity should be feasible and would dramatically improve prostate cancer therapy.
A number of techniques have been investigated to direct therapeutic and diagnostic agents to tumors. These have included targeting of tumor cell surface molecules, targeting regions of activated endothelium, utilizing the dense and leaky vasculature associated with tumors, and taking advantage of the enhanced metabolic and proteolytic activities associated with tumors. Antibody labeling has been used extensively to achieve cell-selective targeting of therapeutic and diagnostic agents. A number of approaches have been taken for antibody-targeting of therapeutic agents. These have included direct conjugation of antibodies to drugs such as interferon-alpha (Ozzello, et al., 1998), tumor necrosis factor (Moro, et al., 1997), and saporin (Sforzini, et al., 1998). Antibody conjugation has also been used for tumor-targeting of radioisotopes for radioimmunotherapy and radioimmunodetection (Zhu, et al., 1998). Currently, there is a commercial product for detection of prostate cancer (ProstaScint) that is an antibody against prostate-specific membrane antigen conjugated to a scintigraphic target (Gregorakis, et al., 1998). Immunoliposomes or affinity liposomes are liposome drug carriers with antibodies conjugated to their surfaces. These drug carriers can be loaded with cytotoxic agents, such as doxorubicin, for destruction of cancerous cells. Antibody targeting is also under investigation for cell-selective gene therapy.
Virus particles have been developed that display single chain antibodies on their surface, allowing specific targeting of a wide variety of cell types (Yang, et al., 1998; Jiang, et al., 1998; Chu and Dornburg, 1997; Somia, et al., 1995). To target regions of activated endothelium, immunoliposomes have been made with antibodies to E-selectin on their surfaces. It may be possible to achieve similar targeting efficiencies with small tumor-specific peptides (Pasqualini, et al., 1997). Recently, tumors have been imaged using protease-activated near-infrared fluorescent probes (Weissleder, (1999). These agents could be administered systemically, were accumulated in the tumors due to the abundant and leaky vasculature, and were activated by the elevated proteolytic enzymes.
The nanoparticles that are the subject of this invention are amenable to these types of targeting methodologies. The nanoparticle surfaces can easily be modified with antibodies, peptides, or other cell-specific moieties. A specific embodiment of these nanoparticles act as absorbers of radiation. These nanoparticles have tunable excitation wavelengths and undergo nonradiative decay back to the ground state by emission of heat. This heat can be used to effect local hyperthermia. Alternatively, these nanoparticles, in addition to acting as absorbers, may scatter light and thereby act as contrast agents as a means to image the local environment in which they reside. Other nanoparticles that are also the subject of this invention are strong visible and infrared fluorophores. Their strong emission is used in imaging applications. It is known that solid metal nanoparticles (i.e. solid, single metal spheres of uniform composition and nanometer dimensions) possess interesting optical properties. In particular, metal nanoparticles display a pronounced optical resonance. Metal nanoparticles are similar to metal colloids in this regard, exhibiting a strong optical absorption due to the collective electronic response of the metal to light. Metal colloids have a variety of useful optical properties including a strong optical absorption and an extremely large and fast third-order nonlinear optical (NLO) polarizability. These optical properties are attributed to the phasic response of electrons in the metallic particles to electromagnetic fields. This collective electron excitation is known as plasmon resonance. At resonance, dilute metal colloid solutions have the largest electronic NLO susceptibility of known substances. However, the utility of these solutions is limited because their plasmon resonance is confined to relatively narrow wavelength ranges and cannot readily be shifted. For example, silver particles 10 nm in diameter absorb light maximally at approximately 355 nm, while similar sized gold particles absorb maximally at about 520 nm. These absorbance maximums are insensitive to changes in particle size and various dielectric coatings on the particles. However, the nanoparticles of this invention are more amenable to a directed shift in their plasmon resonance and hence absorption or scattering wavelengths tan these solid metal nanoparticles.
There have been earlier efforts for therapeutic uses of compositions that emit heat upon excitation, however, these are distinguishable from the present invention. In U.S. Pat. No. 4,983,159, Rand describes the induction of hyperthermia to a neoplasm using particles which exhibit a heating hysteresis when subjected to an alternating magnetic field. However, the particles used in the ""159 patent are more properly described as microparticles and are much larger than the analogous nanoparticles used herein. U.S. Pat. Nos. 4,106,488 and 4,303,636 to Gordon describe particles of nanometer scale dimensions. However, the excitation source is different from that which is used herein and outside the scope of the present invention. As such, it is believed that the underlying physical excitation mechanisms of these earlier works differs from that of the present invention.
A serious practical limitation to realizing many applications of solid metal nanoparticles is the inability to position the plasmon resonance at technologically important wavelengths. For example, solid gold nanoparticles of 10 nm in diameter have a plasmon resonance centered at 520 nm. This plasmon resonance cannot be controllably shifted by more than approximately 30 nanometers by varying the particle diameter or the specific embedding medium.
One method of overcoming this problem is to coat small nonconducting particles with these metals. For example, the reduction of Au on Au2S (reduction of chloroauric acid with sodium sulfide) particles has been shown to red shift the gold colloid absorption maximum from 520 nm to between approximately 600 nm and 900 nm, depending on the amount of gold deposited on the Au2S core and the size of the core. Zhou, et al. (1994). The ratio of the core radius to shell thickness can be controlled by changing the reactant concentrations or by stopping the reaction. In this case, the diameter of the particle core is directly proportional to the red shift in the wavelength of light that induces gold plasmon resonance. However, gold-sulfide particle diameters are limited to sizes of approximately 40-45 nm with a thin gold shell (less than 5 nm). The limited size of the gold-sulfide particles of Zhou et al. limits the absorbance maximum to wavelengths no larger than 900 nm. (Averitt et al. 1997).
An additional limitation of such particles as defined by Zhou et al. is that both the core and the shell are grown as a result of a single chemical reaction, thus limiting the choice of the core material and the shell material to Au2S and Au respectively. In addition, only the ratio of the core radius to shell thickness may be controlled; independent control of the core radius and the shell thickness is not possible.
Nedeljkovic and Patel (1991) disclosed silver-coated silver bromide particles that are produced by intense UV irradiation of a mixture of silver bromide, silver, sodium dodecylsulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). The Neideljkovic particles range in size from approximately 10 to 40 nm and are irregularly shaped, as determined by transmission electron microscopy. Predictably, the spectra obtained from these particle preparations are extremely broad.
U.S. Pat. No. 5,023,139, Birnboim et al. disclosed theoretical calculations indicating that metal-coated, semiconducting, nanometer-sized particles containing should exhibit third-order nonlinear optical susceptibility relative to uncoated dielectric nanoparticles (due to local field enhancement). Their static calculations were based on hypothetical compositions. In those embodiments theoretically proposed by Birnboim et al. that do in fact propose a metal outer shell, there is an additional requirement as to the specific medium in which they must be used in order to properly function.
However, Birnboim does not disclose methods for preparing the disclosed hypothetical compositions. Furthermore, Birnboim""s calculations do not take into account surface electron scattering. Surface electron scattering strongly modifies the optical response of all metallic structures that possess at least one dimension smaller than the bulk electron mean free path (e.g. in Au at room temperature the bulk electron mean free path is about 40 nm). This effect reduces the local field enhancement factor that in turn reduces the resonant third order nonlinear optical susceptibility associated with the nanoshell geometry. See, Averitt et al., 1997. Since typical shell thicknesses for these compositions fall below 40 nm, Birnboim et al""s theoretical calculations fail to account for this effect which is an important aspect of the optical response for functional metal nanoshells.
It is also possible to conduct targeted imaging using fluorescent probes that emit infrared light from an object of interest (e.g., tumor) in vivo (Weissleder, 1999; Pathankar et al., 1997). For imaging, we need to focus on fluorophores and scatterers. Scatterers can be used to drastically change the scattering coefficient (thus acting as an optical contrast agent) in a targeted tissue to allow imaging. Absorbers might potentially be used in this application as well.
It has been discovered that nanoparticles comprising one non-conducting or semiconducting core layer and at least one conducting shell layer, in which the shell layer is independently layered upon said core layer and the thickness of said shell layer is independent of the radius of said core layer, can be manufactured to have the characteristic that the thickness of said shell layer is less than that of a shell layer for which the nanoparticle has a plasmon resonance peak width described by a bulk dielectric function of the material comprising the shell layer. Similarly, these nanoparticles can be manufactured to have plasmon resonance peak widths that are independent of the thickness of the shell layer
Methods and materials have previously been disclosed that can be used to shift the wavelength of maximum resonance of metal nanoparticles called nanoshells. These methods produce materials having defined wavelength absorbance maxima across the visible and infrared range of the electromagnetic spectrum. Particularly, such metal nanoshell composites have been constructed in a manner to allow a choice of core material, core dimensions, and core geometry independent of those criteria for the shell material. Compositions produced by these methods have relatively homogeneous structures and do not have to rely on suspension in a particular medium in order to exhibit their desired absorption characteristics. Of interest herein, these nanoshells overcome the optical limitations of the prior art and which have limited the therapeutic and diagnostic applications discussed above. Such materials were described in U.S. application Ser. No. 09/038,277, filed Apr. 10, 1998; which is specifically and fully incorporated by reference herein.
It is an object of the present invention to provide materials and methods for use in cell and tissue therapy. The primary object is a method for inducing a localized, targeted hyperthermia in such cell and tissue therapy. It is another object of the present invention to provide materials and methods for use in diagnostic imaging.
It is a further object of the present invention to provide methods for using these materials which are minimally invasive and efficacious without systemic side effects.
In the therapeutic embodiment, methods are described in which particles are administered to cells and/or tissue, which upon their exposure to light, effect the in vitro or in vivo, local heating of their immediate environment. In the preferred embodiment, the particles consist of a dielectric or semiconductor core and a conducting shell, the dimension of the particles is on a scale of tens to hundreds of nanometers, and the radiation used is infrared radiation.
In a preferred embodiment, the method is used to treat cancer. In an alternative embodiment, the method is applied to treat non-malignant tumors. In either of these embodiments, the method may be the sole method, or it may be used in combination with another therapy. In another embodiment, the method may be used for cosmetic enhancement.
In a preferred embodiment, the nanoparticle consists of a silica core and a gold shell. In an alternative embodiment, the nanoparticle consist of a gold sulfide core and a gold shell.
In a further embodiment of the general method, the nanoparticles are targeted to a desired location through the use of appropriate chemical schemes. In the preferred embodiment, antigen-antibody binding is used for targeting.
In the diagnostic embodiment, methods are described in which particles are administered to cells and/or tissue, which upon their exposure to radiation, effect the in vitro or in vivo, imaging of their immediate environment. In the preferred embodiment, the particles consist of a dielectric or semiconductive core doped with rare earth ions such as Pr+3, Er+3, and Nd+3, the dimension of the particles is on a scale of tens to hundreds of nanometers, and the radiation used is visible or infrared radiation. Alternatively, the particle may consist of dielectric or semiconductor core and a conducting shell.
In a preferred embodiment, the nanoparticle consists of a silica nanoparticle doped with Pr+3 ions. In an alternative embodiment, the nanoparticle consists of a silica nanoparticle doped with Er+3 or Nd+3. In an alternative embodiment, the nanoparticle consists of a silica core with a gold shell designed as either an absorber or a scatterer.
In both the diagnostic and therapeutic embodiments, the radiation source is preferably electromagnetic radiation, but may alternatively be a non-electromagnetic radiation, such as ultrasound radiation.